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Virology Journal
Published in: Virology Journal 1/2021

Open Access 01-12-2021 | Dengue Virus | Research

Development of genus-specific universal primers for the detection of flaviviruses

Authors: Tomo Daidoji, Ronald Enrique Morales Vargas, Katsuro Hagiwara, Yasuha Arai, Yohei Watanabe, Keisuke Nishioka, Fumi Murakoshi, Kotaro Garan, Hiroki Sadakane, Takaaki Nakaya

Published in: Virology Journal | Issue 1/2021

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Abstract

Background

Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods.

Methods

We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates.

Results

The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates.

Conclusion

This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.
Appendix
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Metadata
Title
Development of genus-specific universal primers for the detection of flaviviruses
Authors
Tomo Daidoji
Ronald Enrique Morales Vargas
Katsuro Hagiwara
Yasuha Arai
Yohei Watanabe
Keisuke Nishioka
Fumi Murakoshi
Kotaro Garan
Hiroki Sadakane
Takaaki Nakaya
Publication date
01-12-2021
Publisher
BioMed Central
Published in
Virology Journal / Issue 1/2021
Electronic ISSN: 1743-422X
DOI
https://doi.org/10.1186/s12985-021-01646-5

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